Okorji, U. P. (2015) Inhibition of Neuroinflammation by Artemisinin and its Derivatives. Doctoral thesis, University of Huddersfield.
- Accepted Version
Restricted to Repository staff only until 28 September 2017.
Available under License Creative Commons Attribution Non-commercial No Derivatives.
Background: Neuroinflammation in the central nervous system involves the rapid reaction of the CNS through activation of resident immune cells (especially microglia cells) to infection, trauma and stroke, among other stimuli. It is a tightly regulated process but in chronic cases it is detrimental. Uncontrolled neuroinflammation has been shown to manifest itself in various neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral sclerosis (ALS) and multiple sclerosis (MS). The Nrf2/HO-1 antioxidant system has also been recently shown to be involved in neuroinflammation. Artemisinin, artemether and artesunate are antimalarial drugs that have been shown to possess anti-inflammatory activity. However, there is no detailed evidence demonstrating that these drugs could inhibit neuroinflammation in lipopolysaccharide (LPS)-activated BV2 microglia cells. Therefore, this study aims to investigate the effects of these compounds in LPS-activated BV2 microglia. This research also attempted to explore the possible involvement of the Nrf2/HO-1 system in the anti-neuroinflammatory action of these compounds.
Methods: Cultured BV2 microglia cells were stimulated with LPS following pre-treatment with artemisinin (5-40 μM), artemether (5-40 μM) or artesunate (0.5-4 μM). Nitrite, used as a measure of nitric oxide production was investigated using the Griess assay. PGE2 production was investigated with PGE2 EIA assay, while cytokine production was measured using ELISA. Reporter gene assays in transfected HEK 293 cells were used to investigate effects of the compounds on NF-B and ARE luciferase activities. Further experiments to elucidate the mechanism of action of artemisinin, artemether and artesunate were carried out by measuring expressions of various target proteins in whole cell and nuclear extracts using western blotting. In addition, NF-B DNA binding was investigated using the EMSA. Subsequent investigations to understand the role of Nrf2 activity on the inhibitory activity of the compounds on NO, TNFα, IL-6 and PGE2 production in activated BV2 cells were carried out in Nrf2 silenced cells. To achieve this, BV2 cells were transiently transfected with Nrf2 siRNA followed by pre-treatment with compounds and stimulation with LPS for 24 hours.
Results: Artemisinin, artemether and artesunate inhibited LPS-induced nitric oxide (NO), prostaglandin E2 (PGE2) and pro-inflammatory cytokine (TNFα and IL-6) production in LPS-activated BV2 microglia cells. These compounds also down regulated the expression of specific regulatory proteins, inducible NO synthase (iNOS) cyclooxygenase-2 (COX-2) and microsomal prostaglandin E2 synthase-1 (mPGES-1).
Artemisinin, artemether and artesunate exerted anti-neuroinflammatory activity in LPS activated microglia by suppressing LPS-induced NF-B activity via inhibition of IB phosphorylation and degradation-mediated nuclear translocation of the p65NF-B subunit. These compounds also produced consistent inhibition of DNA binding of NF-B. All three compounds exhibited a similar profile in interfering with p38MAPK signalling following LPS activation of BV2 microglia by preventing phosphorylation of MKK3/6, p38 and MAPKAPK2. This research showed that artemisinin, artemether and artesunate activated Nrf2 mediated HO-1 antioxidant activity in BV2 cells. These effects appear to be mediated through the activation of the antioxidant response element (ARE). Further association studies revealed that artemisinin; artemether and artesunate caused reduction in TNFα, IL-6 and PGE2 secretion in LPS-activated BV2 cells through Nrf2-dependent mechanism. However, it appeared that their inhibitory action on NO production was independent of Nrf2 activity.
Conclusion: Taken together, the outcome of this research indicates that artemisinin, artemether and artesunate suppress neuroinflammation in LPS-activated BV2 microglia by interfering with NF-B and p38MAPK signalling. The compounds also produced consistent activation of the Nrf2/HO-1 antioxidant protective system. This research has also provided the first evidence that the inhibitory actions of these compounds on TNFα, IL-6 and PGE2 are dependent on their ability to activate the Nrf2 transcription factor.
|Item Type:||Thesis (Doctoral)|
|Subjects:||Q Science > Q Science (General)
Q Science > QD Chemistry
|Schools:||School of Applied Sciences|
|Depositing User:||Elizabeth Boulton|
|Date Deposited:||28 Sep 2015 09:03|
|Last Modified:||25 Aug 2016 17:02|
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